The overall objective of our research is to understand the role of increased spontaneous mutation (mutator phenotypes) in human disease. This exploratory proposal focuses on an understudied but important property of mutators: their inherent instability. Mutator phenotypes fuel oncogenesis by providing the genetic diversity necessary for emergence of malignant clones. However, deleterious mutations also accumulate in mutator cells. Thus, while mutators accelerate oncogenesis, the fitness cost of increased mutation imposes indirect selection pressure to reduce mutation rates. This counter-selection will occur after adaptation to a stable environment where conditions no longer favor the genetic potential of mutators. One possible mechanism to reduce mutation rates is the acquisition of compensatory alleles at modifier loci that suppress the mutator phenotype. To identify genetic pathways that mediate mutator suppression, we developed a suppressor screen in yeast that exploits the synergistic relationship between DNA polymerase (Pol ) proofreading and mismatch repair (MMR). Double mutants are inevitable, suggesting that extreme mutation rates exceed an error threshold. However, variants that escape this error-induced extinction (eex) rapidly emerge from mutator clones. One-third of the escape mutants result from second- site changes in Pol that suppress the mutator phenotype, while two-thirds are due to unknown mutator suppressor alleles in the genome. The goal of the proposed exploratory study is to identify these genomic antimutator alleles and the genes that mediate mutator suppression. We will use two complementary genome-wide approaches: 1) an adaptation of synthetic genetic array (SGA) analysis that allows us to screen all non-essential genes in yeast for their roles in lethal mutagenesis, and 2) a pooled linkage strategy coupled with next-generation sequencing that can specifically identify functional suppressor mutations among the many incidental mutations expected in mutator-derived clones. These experiments promise to break new ground in understanding the fate of eukaryotic mutators by identifying novel genetic interactions and pathways that mediate mutagenesis and repair. The genes discovered in our analysis will provide the foundation for future studies in mice and humans assessing the role of antimutator alleles in the etiology of cancer.